Recently, surface plasmon sensor, localized plasmon sensor, or total reflection fluorescent lighting sensor is used as a sensor for detecting the presence or the extent of biomolecular interaction.
FIG. 1 is a schematic diagram showing a conventional total reflection fluorescent lighting sensor 11. In the total reflection fluorescent lighting sensor 11, a metal thin film 13 such as Au thin film and the like is formed on the upper surface of a transparent substrate 12 made of glass etc., and a great number of acceptors 14 are immobilized on the metal thin film 13. A prism 16 is closely attached to the lower surface of the transparent substrate 12.
The total reflection fluorescent illumination sensor 11 is arranged so that the acceptors 14 directly contact a flow path 17 through which the analysis sample solution flows. The ligand 15 in the analysis sample solution is modified with fluorescent molecules in advance. The excitation light exits from a light source 18 towards the prism 16 so that an incident angle at the interface between the transparent substrate 12 and the metal thin film 13 becomes an angle greater than the total reflection angle at the relevant interface. The excitation light that has passed the prism 16 and the transparent substrate 12 is totally reflected at the interface of the metal thin film 13 and the transparent substrate 12. The evanescent light is then generated at the back surface of the metal thin film 13, and the electric field of the evanescent light transmits through the metal thin film 13 and the acceptors 14 and spreads. Furthermore, a greater electric field is generated by the surface plasmon in the metal thin film 13 excited by the evanescent light. The electric field generated by the evanescent light and the surface plasmon excites the fluorescent molecules of the ligand 15 bonded to the acceptor 14 and light is emitted. The presence of a specific ligand 15 bonded to the acceptor 14, or the amount of the ligand 15 bonded to the acceptor 14 can be measured by measuring the emission intensity by means of a light detector 19 arranged facing the acceptor 14.
However, since the electric field generated by the evanescent light and the like diffuses up to the distance of 200 to 300 nm from the surface of the transparent substrate 12, as shown in FIG. 1, not only the ligand 15 bonded to the acceptor 14, but even the fluorescent molecules of the ligand 15 not bonded to the acceptor 14 are excited, which becomes a noise in the measured data. Particularly, the noise becomes larger the higher the concentration of the ligand 15 in the analysis sample solution. The noise becomes the cause of significant lowering in the measurement accuracy of the total reflection fluorescent illumination sensor since the size of the biomolecules is a several tens of nm, and thus the detection accuracy of about one molecule of analyte is difficult to obtain.
FIG. 2 is a schematic diagram showing a conventional localized plasmon resonance sensor 21 (patent article 3). In the localized plasmon resonance sensor 21, a great number of metal fine particles 23 of Au and the like having a diameter of 10 to 20 nm are fixed on one surface of the transparent substrate 22 made of glass etc. to configure a sensor unit 24. The light beam is irradiated perpendicular to the sensor unit 24 from the light source 25 on the side opposite the surface fixed with the metal fine particles 23, and the absorption spectrum of the light that has transmitted through the metal fine particles 23 is measured with a spectrophotometer 26 to obtain the absorbance. A strong absorption peak appears for the light near the wavelength of 520 nm in such localized plasmon resonance sensor 21.
In the localized plasmon resonance sensor 21, the change in index of refraction at the vicinity of the metal fine particles can be detected from the change in absorbance. As shown in FIG. 3, when the acceptor 27 is immobilized to the surface of the metal fine particles 23 fixed to the transparent substrate 22 of the sensor unit 24, the presence or the amount of the specific ligand 28 can be detected since the index of refraction at the periphery of the metal fine particles 23 changes and the absorbance of the light that has transmitted through the metal fine particles 23 changes if a specific ligand 28 is attached to the acceptor 27.
In such localized plasmon resonance sensor, the prism as in the total reflection fluorescent illumination sensor is not necessary and miniaturization is possible since the absorptivity of the transmitted light that has transmitted through the metal fine particles is being measured. Furthermore, when the metal fine particles are used, only the change in the vicinity of the metal fine particles can be detected since the electric field localizes as opposed to the metal thin film (total reflection fluorescent illumination sensor), whereby measurement of the analyte in a small region becomes possible and the influence of the analyte at a location distant from the metal fine particles can be reduced.
However, the change in index of refraction is very small according to this method, and thus the change in absorbance is also very small. Therefore, the detection accuracy of about one molecule is difficult to obtain even with such localized plasmon resonance sensor.    [Patent article 1] Japanese Laid-Open Patent Publication No. 2000-131237    [Patent article 2] Japanese Patent No. 3452837    [Patent article 3] Japanese Laid-Open Patent Publication No. 6-27023